肿瘤相关成纤维细胞中微小RNA-214-3p表达对卵巢癌细胞顺铂敏感性的影响

Effect of microRNA-214-3p expression in cancer-associated fibroblasts on cisplatin sensitivity of ovarian cancer cells

目的探讨肿瘤相关成纤维细胞(CAFs)中微小RNA-214-3p(miR-214-3p)表达对卵巢癌细胞顺铂敏感性的影响及其作用机制。方法选取64例卵巢癌患者作为研究对象,根据化疗后无进展生存期分为铂部分敏感组和铂敏感组,采用实时荧光定量聚合酶链反应(qRT-PCR)检测2组患者卵巢癌组织中miR-214-3p相对表达量,比较不同临床特征患者的2年生存率;原代培养CAFs及正常卵巢成纤维细胞(NFs),采用qRT-PCR、免疫荧光实验检测CAFs和NFs中miR-214-3p、p62蛋白表达;通过CSIOVDB数据库检索SQSTM1基因在不同种类卵巢细胞中的表达水平;向CAFs瞬时转染miR-214-3p mimic(mimic组)和miR-214-3p mimic NC(NC组),将未转染CAFs设为对照组;留取各组细胞培养上清,建立卵巢癌细胞SKOV3与各组CAFs间接共培养模型,采用CCK-8法、DCFH-DA法、qRT-PCR及免疫印迹法分别检测不同培养条件下SKOV3细胞增殖率、顺铂半数抑制浓度(IC 50)、细胞活性氧(ROS)含量和miR-214-3p、顺铂耐药基因CCND1、自噬蛋白p62相对表达量。结果铂部分敏感组患者的miR-214-3p相对表达量低于铂敏感组,差异有统计学意义(P<0.01);不同国际妇产科联盟(FIGO)分期、铂部分敏感情况、miR-214-3p低表达情况患者的2年生存率比较,差异有统计学意义(P<0.01);卵巢CAFs中miR-214-3p相对表达量低于NFs,p62蛋白表达水平高于NFs,差异有统计学意义(P<0.01);CSIOVDB数据库在线分析显示,卵巢癌CAFs中的SQSTM1基因表达水平高于卵巢癌上皮细胞、NFs,差异有统计学意义(P<0.01);相较于与对照组CAFs、NC组CAFs间接共培养的SKOV3细胞,与mimic组CAFs间接共培养的SKOV3细胞的增殖率、顺铂IC 50、ROS含量降低,miR-214-3p相对表达量升高,CCND1mRNA和p62蛋白相对表达量降低,差异有统计学意义(P<0.01)。结论CAFs中miR-214-3p表达与卵巢癌细胞对顺铂的敏感性相关,CAFs中miR-214-3p低表达可促进卵巢癌细胞增殖及ROS介导的自噬,进而降低卵巢癌细胞对顺铂的敏感性。

Objective To investigate the effect of microRNA-214-3p(miR-214-3p)expression in cancer-associated fibroblasts(CAFs)on the cisplatin sensitivity of ovarian cancer cells and its mechanism.Methods Sixty-four ovarian cancer patients were selected as study subjects and divided into platinum-partially sensitive group and platinum-sensitive group based on progression-free survival after chemotherapy.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the relative expression of miR-214-3p in ovarian cancer tissues from the two groups,and the 2-year survival rates of patients with different clinical characteristics were compared.CAFs and normal ovarian fibroblasts(NFs)were primarily cultured,and qRT-PCR and immunofluorescence experiments were used to detect the expression of miR-214-3p and p62 protein in CAFs and NFs.The expression levels of SQSTMI gene in different types of ovarian cells were searched through the CSIOVDB database.CAFs were transiently transfected with miR-214-3p mimic(mimic group)and miR-214-3p mimic NC(NC group),and untransfected CAFs were selected as control group.Cell culture supernatants from each group were collected,and an indirect co-culture model of ovarian cancer cell line SKOV3 with CAFs from each group was established.The CCK-8 method,DCFH-DA method,qRTPCR,and immunoblotting were used to detect the proliferation rate,half-maximal inhibitory concentration of cisplatin(IC50),reactive oxygen species(ROS)content,and relative expression levels of miR-214-3p,cisplatin resistance gene CCNDI,and autophagy protein p62 in SKOV3 cells under different culture conditions.Results The relative expression of miR-214-3p was lower in the platinum-partially sensitive group than in the platinum-sensitive group(P<0.01).There were statistically significant differences in the 2-year survival rates among patients with different International Federation of Gynecology and Obstetrics(FIGO)stages,platinum partial sensitivity,and low miR-214-3p expression(P<0.01).The relative expression of miR-214-3p was lower in ovarian CAFs than in NFs,while the expression level of p62 protein was higher in CAFs than in NFs(P<0.01).Online analysis of the CSIOVDB database showed that the expression level of SQSTMI gene in ovarian CAFs was higher than that in ovarian cancer epithelial cells and NFs(P<0.01).Compared with SKOV3 cells indirectly co-cultured with CAFs in the control group and CAFs in NC group,SKOV3 cells indirectly co-cultured with mimic CAFs showed reduced proliferation rate,cisplatin IC50,and ROS content,increased relative expression of miR-214-3p,and decreased relative expression of CCND1 mRNA and p62 protein(P<0.01).Conclusion The expression of miR-214-3p in CAFs is correlated with the sensitivity of ovarian cancer cells to cisplatin.Low expression of miR-214-3p in CAFs can promote the proliferation of ovarian cancer cells and ROS-mediated autophagy,thereby reducing the sensitivity of ovarian cancer cells to cisplatin.